PPE Export by the Essential ESX-5 Secretion System in M. tuberculosis Virulence

项目摘要/摘要 结核分枝杆菌(Mtb)的体外生长需要ESX-5型VII蛋白分泌系统。 因为ESX-5是必不可少的，ESX-5在结核分枝杆菌毒力和通过ESX-5输出的蛋白质中所起的作用 支持结核分枝杆菌复制的基因特征仍然很差。在相关病原体海洋分枝杆菌中， ESX-5输出许多分枝杆菌所特有的PE和PPE蛋白家族的蛋白质。几个 MTB PE和PPE蛋白定位于细胞壁，最近被认为与获得不同的 营养素。流行的PE和PPE分泌模型表明，它们以成对的异源二聚体的形式输出 唯一，且该导出由针对PE-PPE对同源ESX的ESPG伴侣来促进 用于出口的分泌物系统。这一提议将检验以下中心假设：MTB需要ESX-5活动，并且 ESX-5底物的一个子集，它独立于EspG5分子伴侣输出以获得毒力。这 假设是基于使用结核分枝杆菌菌株的初步数据，在这些菌株中，ESX-5的分泌机制EccD5 亚单位，或EspG5伴侣可以有条件地耗尽。EcD5但不是EspG5限制的Mtb耗尽 在巨噬细胞和某些碳源上生长。具体目标1将确定ESX-5的作用和 EspG5伴侣蛋白在结核分枝杆菌毒力中的作用 小鼠气溶胶感染模型。特殊目标2将使用ESX-5条件耗竭菌株来鉴定PPE 需要ESX-5活性但不需要EspG5伴侣的蛋白质用于出口并连接ESX-5活性以使用 特定的营养素。具体目标3将检查PE蛋白之间的功能冗余，以促进 利用小鼠气溶胶感染模型输出某些PPE蛋白并促进结核分枝杆菌毒力。这个 拟议的研究在概念上是创新的，因为它挑战了现有的ESX-5基片出口模型 通过暗示PE和PPE蛋白之间的混杂配对和某些PPE蛋白的出口 独立于EspG5的伴侣。这项拟议的研究意义重大，因为它有望表明 MTB的发病机制需要ESX-5活性。这一知识将使ESX-5成为 开发新的抗结核药物并支持将ESX-5分泌的抗原纳入新的 结核病疫苗。

PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) requires the ESX-5 Type VII protein secretion system for in vitro growth. Because ESX-5 is essential, the role that ESX-5 plays in Mtb virulence and the proteins exported via ESX-5 that support Mtb replication remain poorly characterized. In the related pathogen Mycobacterium marinum, ESX-5 exports many proteins of the PE and PPE protein families that are unique to the mycobacteria. Several Mtb PE and PPE proteins localize to the cell wall and were recently implicated in acquisition of diverse nutrients. The prevailing model of PE and PPE secretion suggests they are exported as heterodimers that pair uniquely and that export is promoted by an EspG chaperone that targets the PE-PPE pair to the cognate ESX secretion system for export. This proposal will test the central hypothesis that Mtb requires ESX-5 activity and a subset of ESX-5 substrates that are exported independently of the EspG5 chaperone for virulence. This hypothesis is based on preliminary data using Mtb strains in which EccD5, an ESX-5 secretion machinery subunit, or the EspG5 chaperone can be conditionally depleted. Depletion of EccD5 but not EspG5 limited Mtb growth in macrophages and on certain carbon sources. Specific Aim 1 will determine the role of ESX-5 and the EspG5 chaperone in Mtb virulence using conditional depletion of EccD5 or EspG5 and both macrophage and mouse aerosol infection models. Specific Aim 2 will use the ESX-5 conditional depletion strains to identify PPE proteins that require ESX-5 activity but not the EspG5 chaperone for export and connect ESX-5 activity to use of specific nutrients. Specific Aim 3 will examine functional redundancy between PE proteins in facilitating export of certain PPE proteins and promoting Mtb virulence using the mouse aerosol infection model. The proposed research is conceptually innovative because it challenges existing models of ESX-5 substrate export by suggesting promiscuous pairing between PE and PPE proteins and export of certain PPE proteins independent of the EspG5 chaperone. The proposed research is significant because it is expected to show that Mtb requires ESX-5 activity for pathogenesis. This knowledge would establish ESX-5 as a strategic target for development of new anti-tubercular drugs and support inclusion of ESX-5-secreted antigens in new tuberculosis vaccines.